Dynein was first discovered as the microtubule-based motor that powers the movement of cilia and flagella. Subsequently, a cytoplasmic form of dynein was found to move numerous cargos (membrane organelles, the nucleus, mRNAs, proteins, microtubules, and viruses) towards the microtubule minus end in most eukaryotic cells. Mutations in dynein or its regulatory proteins have been associated with congenital defects (e.g. situs inversus, lissencephaly), and modulating dynein transport may provide new strategies for treating viral infections, cancer, and neurodegenerative disease. Despite its importance in cell biology and medicine, dynein-based motility is poorly understood in comparison to kinesin and myosin, the two other major cytoskeletal motor proteins. A major deficiency in the dynein field is the lack of atomic resolution structural information of its motor domain, and crystallization has been difficult to achieve because its very large size (>300 kDa). In this grant application, we propose to obtain the first crystal structure of the motor domain of yeast cytoplasmic dynein. By crystallizing the motor in different nucleotide states, we also will seek to obtain "snap shots" of dynein in different stages of its ATPase cycle. These X-ray crystallography studies will be complemented by single molecule motility studies to test how dynein produces motility. Based upon our crystal structure, we will design new recombinant dyneins that will enable placement of fluorescent dyes and other biophysical probes in defined locations on the dynein motor. Using such probes, we will measure by nucleotide-dependent conformational changes of the motor using single molecule assays and test whether they are important for motility. We also will address the role of dynein's 4 ATP binding and determine whether dynein uses one or multiple ATPs when it takes a step. Finally, we will embark on the first in vitro motility studies of a second class of cytoplasmic dyneins involved in transporting proteins from the tips of cilia/flagella to the cell body. In summary, these studies will provide new information on dynein's structure, how it utilizes nucleotides and changes its conformation, and how it has adapted for unique transport functions in the cytoplasm and the flagellum. The reagents and structures generated in this work will be broadly valuable to the entire dynein field. Dynein is a member of the AAA+ ATPase superfamily, and thus results of our studies will be valuable for understanding this large family of ATPases. Our structural studies also will provide new ideas on how dynein can be regulated by cellular regulatory proteins as well as potentially by therapeutic drugs. PUBLIC HEALTH RELEVANCE: Dynein, a microtubule-based motor protein that powers cilary beating and the intracellular transport of membranes, proteins and mRNAs, is involved in cellular processes that when defective can give rise to brain abnormalities, heart defects, kidney malfunction, immotile sperm and neurodegenerative disease. Dynein remains poorly understood in comparison to other motors such as kinesin and myosin, in part due to the absence of an atomic structure for its motor domain. In this grant, we propose to solve x-ray crystal structures of the motor domain and to develop new single molecule motility assays for dynein;the results of such studies will lead to new models for dynein-based motility and for how dynein might be regulated by cellular proteins or possibly small molecule drugs.